KINETIC, DYNAMIC, AND PATHWAY STUDIES OF GLYCEROL METABOLISM BY KLEBSIELLA-PNEUMONIAE IN ANAEROBIC CONTINUOUS-CULTURE - IV - ENZYMES AND FLUXES OF PYRUVATE METABOLISM

The activities of pyruvate kinase (PK), pyruvate: formate-lyase (PFL), pyruvate dehydrogenase (PDH), and citrate synthase (CS) involved in t he anaerobic glycerol conversion by Klebsiella pneumoniae were studied in continuous culture under conditions of steady states and sustained oscillations. Both the in vitro and in vivo activities of PK, PFL, an d PDH are strongly affected by the substrate concentration and its upt ake rate, as is the in vitro activity of CS. The flux from phosphoenol pyruvate to pyruvate is found to be mainly regulated on a genetic leve l by the synthesis rate of PK, particularly at low substrate concentra tion and low growth rate. In contrast, the conversion of pyruvate to a cetyl-CoA is mainly regulated on a metabolic level by the in vivo acti vities of PFL and PDH. The ratio of in vitro to in vivo activities is in the range of 1 to 1.5 for PK, 5 to 17 for PFL and 5 to 80 for PDH u nder the experimental conditions. The regulation of in vivo activity a nd synthesis of these enzymes Is sensitive to fluctuations of culture conditions, leading to oscillations of both the in vitro and in vivo a ctivities. In particular, PFL is strongly affected during oscillations ; its average in vitro activity is only about half of its correspondin g steady-state value under similar environmental conditions. The avera ge In vitro activities of PDH and PK under oscillations are close to t heir corresponding steady-state values. In contrast to all other enzym es measured for the glycerol metabolism by K. pneumoniae PFL and PDH a re more effectively in vivo utilized under oscillations than under ste ady state, underlining the peculiar role of pyruvate metabolism in the dynamic responses of the culture. (C) 1998 John Wiley & Sons, Inc.